To learn whether either reduced de novo cholesterol synthesis and/or altered cholesteryl ester metabolism is responsible for the deficient progestin production induced by estrogen withdrawal from pseudopregnant rabbits, we measured the luteal activity of three enzymes: 1) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (the rate-limiting step in de novo cholesterol synthesis), 2) cholesteryl ester hydrolase, and 3) acyl coenzyme A:cholesterol acyltransferase (ACAT) in estrogen-stimulated and estrogen-deprived rabbits. The only change in the activity of these enzymes and of the enzyme NADPH-cytochrome c reductase (a microsomal marker enzyme) after estrogen capsule removal for 12 or 24 h was a 30% decrease in HMG-CoA reductase activity after 24 h. The decrease in HMG-CoA reductase activity was not accompanied by a detectable change in either the content or localization of cellular free cholesterol. Previous data from our laboratory have demonstrated that 24 h of estrogen deprivation has no effect on inner mitochondrial membrane P-450 side-chain cleavage activity (a rate-limiting step in the conversion of cholesterol to steroid hormones). These data, and our earlier finding that estrogen deprivation leads to accumulation of cholesteryl ester in the luteal cells, indicate that estrogen maintains rabbit luteal progestin production by stimulating the transfer of cytoplasmic cholesterol to the active site of P-450 side-chain cleavage on the inner mitochondrial membrane.